1. Field of the Invention
The present invention relates to the identification, characterization and sequencing of a gene, the mugA gene, and to a live, attenuated strain of V. anguillarum used as a vaccine against vibriosis.
2. Description on of the Related Art
Vibriosis is one of the major bacterial diseases affecting fish, bivalves and crustaceans in the marine environment. Vibrio anguillarum, a gram negative curved rod, is the causative agent of vibriosis. Symptoms of the disease in salmonids include red necrotic lesions in the abdominal muscle, erythema at the base of the fins, and subdermal hemorrhaging. The rectum becomes distended and filled with fluid, and hemorrhaging can often be observed in the internal organs. The bacteria become dispersed throughout the host tissues, including the kidney, liver and spleen. The highest concentration of bacteria is often found in the blood. Vibriosis typically culminates in a hemorrhagic septicemia that causes infected stocks to suffer mortalities ranging from 30-100%. As a result, vibriosis is a significant limitation to aquaculture, causing large economic losses to the industry.
The fish gastrointestinal tract has been implicated as a site of colonization and growth of pathogenic Vibrio species. (Home et al.; J. Fish Dis. 6: 461-471 (1983). It has been discovered that Vibrio ordalii and Vibrio anguillarum primarily infect the intestinal tracts and pyloric cecae of Pacific salmon. (Ransom et al.; J. Fish Dis. 7 107-115 (1984). It has been suggested that the fish gastrointestinal tract serves as a portal entry for V. anguillarum, and that the infection of the fish host begins with the colonization of the posterior gastrointestinal tract and the rectum. (Olsson et al.; J. Fish Dis. 19:225-234 (1996). It has been demonstrated that V.anguillarum exhibits chemotactic mobility towards intestinal mucus, and that motile V. anguillarum cells penetrate crude mucus preparations. Additionally, it has been demonstrated that V. anguillarum adheres to fish intestinal tissue and mucus and to the brush border cells of larval turbot. (Bordas et al.; Appl. Environ. Microbiol. 62:3650-3654 (1996)(Grisez et al.; Dis. Aquat. Org. 26: 181-187 (1996). Following infection of the gastrointestinal tract, it appears that V. anguillarum cells traverse the intestinal epithelium, enter the lamina propria, and spread systemically.
Gastrointestinal mucus is a rich nutrient source that many organisms, including pathogens, can utilize for growth. Studies suggest that growth in mucus is a critical factor to intestinal colonization of a host by pathogens, and that specific physiological changes occur in these bacteria in response to growth in mucus. (Burghoff et al.; Infect. Immun. 61:1293-1300 (1993)(Krivan et al.; Infect. Immun. 60: 3943-3946 (1992). It has been demonstrated that V. anguillarum grows rapidly and efficiently in salmon intestinal mucus. (Garica et al.; Appl. Environ. Microbiol. 63: 1034-1039 (1997). The cells typically exhibit a rapid generation time and express at least five new membrane proteins in mucus-salts medium. Four of the proteins produced during growth in mucus are located in the outer membrane of the cell, while the fifth is located in the cytoplasmic membrane. (Garica et al.; Appl. Environ. Microbiol. 63: 1034-1039 (1997). The present invention provides vaccines and methods for making the same that can be used to prevent vibriosis infection in animals.